In many cases, a compound used as a medicament interacts with a protein in a living body so as to exert its functions. For example, an enzyme inhibitor may bind to a target enzyme so as to inhibit the enzyme reaction, thereby changing biochemical reactions in a living body and expressing its pharmacological action. Thus, in order to search for a medicament, it is important to understand the binding strength of a protein with a candidate compound for such a medicament.
In recent years, as a means for analyzing the interactions of proteins with chemical substances, a surface plasmon resonance method has become a focus of attention. This method comprises immobilizing a protein on a metal surface such as gold, adding a chemical substance to the metal surface, and measuring the change in the surface plasmon resonance, so as to quantify the interaction of the protein with the chemical substance.
A test substance that is a chemical substance is adsorbed to a molecule interacting therewith (that is, a protein or the like immobilized on a metal surface) over time. The amount of the test substance adsorbed, that is, the change in SPR signals, is changed according to the following formula (1):dR/dt=ka·Cs·(Rmax−R)−kd·R   Formula (1)wherein, in the formula (1), R represents an SPR signal; Rmax represents the maximum SPR signal; ka represents an adsorption rate constant; kd represents a dissociation rate constant; and Cs represents a concentration of a test substance located adjacent to the metal surface.
Herein, the value of Cs is constant under ideal conditions where the liquid on the metal surface is constantly substituted with a fresh liquid. Thus, it is possible to determine ka and kd from the measurement results by solving a simple differential equation. A binding constant (KD) indicates the ratio between adsorption and dissociation, and it is represented by kd/ka.
Regarding such surface adsorption, when the n types of multiple compounds are adsorbed to a single adsorption site, the structure of which is not changed by adsorption, since such compounds are competitively adsorbed thereto, a change in SPR signals is represented by the following formula:
                                          ⅆ            R                                ⅆ            t                          =                  A          ·                                    ∑                              i                =                1                            n                        ⁢                                          (                                                                            Ka                      i                                        ·                                          Cs                      i                                        ·                                          (                                              1                        -                                                                              ∑                                                          i                              =                              1                                                        n                                                    ⁢                                                      θ                            i                                                                                              )                                                        -                                                            kd                      i                                        ·                                          θ                      i                                                                      )                            ·                              MW                i                                                                        Formula        ⁢                                  ⁢                  (          2          )                    wherein, in the formula (2), A represents a constant used for conversion from the adsorbed weight to SPR signals; and MWi, Csi, θi, kai, and kdi represent the molecular weight of compound i, the concentration thereof, the adsorption site share thereof, an adsorption rate constant, and a dissociation rate constant, respectively.
Herein, if ka and kd have previously been known, a change in SPR signals obtained when the n types of multiple compounds are adsorbed can be calculated by formula (2).
In such surface absorption, a change in SPR signals obtained when the n types of multiple compounds are adsorbed to different adsorption sites is represented by the following formula (3):
                                          ⅆ            R                                ⅆ            t                          =                              ∑                          i              =              1                        n                    ⁢                      (                                                            Ka                  i                                ·                                  Cs                  i                                ·                                  (                                      R                    ⁢                                                                                  ⁢                                                                  max                        i                                            ⁢                                              -                                                                              ∑                                                          i                              =                              1                                                        n                                                    ⁢                                                      R                            i                                                                                                                                )                                            -                                                kd                  i                                ·                                  R                  i                                                      )                                              Formula        ⁢                                  ⁢                  (          3          )                    wherein, in the formula (3), Ri represents a change in SPR signals of compound i; and Rmaxi represents the maximum adsorbed SPR signals of compound i.
Herein, if ka and kd have previously been known, a change in SPR signals obtained when the n types of multiple compounds are adsorbed can be calculated by formula (3).
It has been known that many physiologically active substances that become targets of medicaments change their structures when these substances bind to certain substances, so that they come to bind to other substances. For example, a single-stranded DNA forms a double strand together with DNA complementary thereto, so that it comes to bind to a certain type of DNA-recognition protein. In addition, it has also been known that a medicament acting on a certain type of enzyme (for example, cytokine, hormone, etc.) binds to a site of the enzyme known as an allosteric site that is different from a substrate recognition site, so that it changes the bindability of the substrate recognition site. Moreover, when a single protein constituting a complex protein is used by itself, it does not have the ability to recognize a physiologically active substance. However, when a plurality of such proteins form a complex, the complex protein can recognize a physiologically active substance.
Thus, for the development of medicaments, it is important to understand a phenomenon whereby multiple substances are simultaneously adsorbed to a ligand and to analyze a test substance by a means for evaluating such adsorption properties. However, the conventional adsorption analysis method only enables determination by competitive adsorption. For example, JP Patent Publication (Kokoku) No. 7-111435 B (1995) describes the analysis of multiple compounds to be analyzed binding to a sensor surface. Chem. Lett. 2002(3), 342 describes that a molecule to be analyzed is allowed to competitively bind to the surface, that it is compared with a case where no molecules to be analyzed are present, and that the concentration of the molecule to be analyzed is detected based on the fact that the change in SPR signals decreases as the above concentration decreases.
In order to search for medicaments, screening involving 1,000,000 or more types of compounds has commonly been conducted. It has been obvious that more experiments are required for the analysis of complex actions. Nevertheless, there have been no methods for differentiating competitive adsorption from a change in an adsorption site itself, so as to process a large amount of data rapidly. Accordingly, it is impossible to exhaustively study such complex actions under current circumstances.